This invention relates generally to the fields of organic chemistry and biological assays. More specifically, the invention relates to methods and compositions for determining optimal antisense sequences, and optimized libraries of oligonucleotide analogs.
Antisense technology is based on the finding that DNA and/or RNA transcription or translation can be modulated using an oligonucleotide which binds to the target nucleic acid. By exploiting the Watson-Crick base pairing, one can design antisense molecules having a very high degree of specificity for the target nucleic acid. Oligonucleotides having only standard (xe2x80x9cnaturalxe2x80x9d) bases and backbones must in general contain at least 17 bases in order to bind with sufficient energy to effectively down-regulate gene expression by activating RNase H.
However, even given DNA or RNA of known sequence, it is still difficult to design an optimally effective antisense molecule. This is because nucleic acids are subject to the formation of a variety of secondary and tertiary structures in vivo, and are frequently coiled, supercoiled, folded, and/or obscured by proteins. Some portions of the target sequence are much more susceptible to binding and hybridization by antisense molecules, while other portions of the target sequence are essentially hidden or unavailable. Typically, 20 to 50 oligonucleotides are tested to find one or more active antisense sites per gene.
Standard methods for selecting antisense sites within pre-mRNA or mRNA sequences are insufficient for the rapid, high-throughput application of antisense to large scale target validation programs. Oligonucleotides must be xe2x80x9ccustom-synthesizedxe2x80x9d for each target site within each target gene. A standing library of millions or billions of conventional oligonucleotides would be required to successfully target each of the approximately 100,000 human genes. An ordered library of millions of antisense oligonucleotides is beyond the chemical, physical, and organizational tools currently available.
A new method for preparing and testing antisense and ribozyme sequences has now been invented.
One aspect of the invention is a composition comprising two oligonucleotide analogs, each having a binding domain and a coupling moiety, wherein the binding domains are capable of hybridizing to a target polynucleotide, and the coupling moieties are capable of coupling to each other in the absence of a target molecule.
Another aspect of the invention is a compound of the formula R1xe2x80x94L1xe2x80x94Xxe2x80x94Axe2x80x94Yxe2x80x94L2xe2x80x94R2, wherein R1 is an oligonucleotide, or an oligonucleotide analog, capable of binding to RNA; R2 is an oligonucleotide, or an oligonucleotide analog, capable of binding to RNA; L1 and L2 are each independently a linking moiety or a bond; X and Y are each independently a coupling moiety; and A comprises a link selected from the group consisting of a covalent bond, a metal ion, and a non-covalent bond, wherein said compound is capable of activating a nuclease or catalyzing cleavage when bound to a target polynucleotide.
Another aspect of the invention is a method for cleaving a target polynucleotide, comprising providing a target RNA molecule; contacting the target RNA molecule with a first oligonucleotide analog, comprising a first binding domain capable of binding a first region of a target polynucleotide, and a first coupling moiety capable of binding to a second coupling moiety, and a second oligonucleotide analog, comprising a second binding domain capable of binding a second region of said target polynucleotide, and a second coupling moiety capable of binding to said first coupling moiety, wherein said first and second binding domains are capable of binding simultaneously to said target RNA molecule; and incubating said target RNA molecule, first analog and second analog in the presence of an RNase capable of cleaving the RNA target.
Another aspect of the invention is a method for cleaving a target RNA molecule, comprising providing a target RNA molecule; contacting the target RNA molecule with a first oligonucleotide analog, comprising a first binding domain capable of binding a first region of a target polynucleotide, and a first coupling moiety capable of binding to a second coupling moiety, and a second oligonucleotide analog, comprising a second binding domain capable of binding a second region of said target polynucleotide, and a second coupling moiety capable of binding to said first coupling moiety, wherein said first and second binding domains are capable of binding simultaneously to said target RNA molecule; and incubating said target RNA molecule, first analog and second analog in the presence of an RNase capable of cleaving the RNA target.
Another aspect of the invention is an antisense library, comprising a set of first oligonucleotide analogs, each first analog comprising a first coupling moiety and a first binding domain, said first binding domain comprising a first backbone and a plurality of first bases capable of base-pairing with a target nucleic acid; and a set of second oligonucleotide analogs, each second analog comprising a second coupling moiety capable of coupling specifically to said first coupling moiety, and a second binding domain, said second binding domain comprising a second backbone and a plurality of second bases capable of base-pairing with a target nucleic acid; wherein an antisense analog consisting of a first analog coupled to a second analog is capable of binding to a target nucleic acid and serving as a nuclease substrate.
Another aspect of the invention is a library of antisense precursor compounds, a plurality of compounds of formula 2 (R1xe2x80x94L1xe2x80x94X) and a plurality of compounds of formula 3 (Yxe2x80x94L2xe2x80x94R2), wherein R1 and R2 are each independently an oligonucleotide or an oligonucleotide analog, capable of binding to mRNA; L1 and L2 are each independently a linking moiety or a bond; X and Y are each independently a coupling moiety; and wherein said compounds of formula 2 and formula 3 can be coupled to form a compound capable of recruiting or activating a nuclease when bound to a target polynucleotide.
Another aspect of the invention is a library of ribozyme precursor compounds, comprising a plurality of compounds of formula 4 (GG-R1-CUGAUGA-L1-X) and a plurality of compounds of formula 5 (Y-L2-GAA-R2), wherein R1 and R2 are each independently an oligonucleotide or an oligonucleotide analog, capable of binding to RNA; L1 and L2 are each independently a linking moiety or a bond; X and Y are each independently a coupling moiety; and wherein said compounds of formula 4 and formula 5 can be coupled to form a ribozyme.
Another aspect of the invention is a method for determining an optimal antisense site for a given mRNA, comprising: selecting a plurality of first oligonucleotide analogs, said first analogs comprising a first coupling moiety and a first binding domain which is complementary to said mRNA; selecting a second oligonucleotide analog for each first oligonucleotide analog, said second analog comprising a second coupling moiety capable of binding said first coupling moiety, and a second binding domain which is complementary to said RNA at a position proximal to the site to which said first binding domain is complementary; coupling said first coupling moieties and said second moieties to provide a plurality of antisense probes; contacting said mRNA with said antisense probes in the presence of an RNase to form a cleavage product; and determining which antisense probe corresponds to said cleavage product.
Another aspect of the invention is a method for determining an optimal ribozyme cleavage site for a given target RNA, comprising: selecting a plurality of first oligonucleotide analogs, said first analogs comprising a first coupling moiety and a first binding domain which is complementary to said target RNA; selecting a second oligonucleotide analog for each first oligonucleotide analog, said second analog comprising a second coupling moiety capable of binding said first coupling moiety, and a second binding domain which is complementary to said RNA at a position proximal to the site to which said first binding domain is complementary; coupling said first coupling moieties and said second moieties to provide a plurality of ribozymes; contacting said target RNA with said ribozymes to form a cleavage product; and determining which ribozyme corresponds to said cleavage product.
One object of the invention is to provide a method for preparing antisense or ribozyme molecules quickly, using a feasible number of pre-synthesized components.
Another object of the invention is to provide a library of components suitable for forming antisense or ribozyme molecules on demand.
Another object of the invention is to provide methods for determining the optimal antisense or ribozyme sequence for a given target.
Another object of the invention is to provide methods for determining an antisense or ribozyme sequence when the target polynucleotide sequence is unknown.